The overall objective of the proposed research is to examine the asymmetric transbilayer distribution of sterols across mammalian cell membranes. The long-term goal of this proposal is to elucidate the structural, the functional, and the physiological consequences of perturbation of the asymmetric distribution of cholesterol across cell membranes so as to provide a better understanding of the fundamental mechanism whereby cell membrane functions are modulated by transbilayer sterol distribution. Specifically, we have chosen as model systems two cell types, red blood cells and LM fibroblasts, since their plasma membrane transbilayer sterol distributions are opposite. Specifically, the effects of lipid manipulation (cholesterol content, fatty acid composition, and phospholipid polar head group composition) and other factors (protease, neuraminidase, aging, soluble proteins, and drug binding) on the asymmetric distribution of sterol across the plasma membranes of these dissimilar cell types will be determined. Fluorescent sterol analogues and a new technique for measuring sterol asymmetry, exchange, and flip-flop will be utilized. The fluorescent sterols will be incorporated into cells, membrane vesicles, and/or phagosomes. At appropriate intervals during the in vitro manipulations plasma membranes will be isolated and the following parameters will be evaluated: (1) Lipid composition; (2) Structure of the membrane and transbilayer distribution of sterols and aminophospholipids; (3) Metabolic interrelationships; and (4) Function: The physiological role of an asymmetric distribution of sterol in membranes will be determined by examination of transmembrane processes that should be affected by sterol content. The significance of the proposed investigation is that basic, fundamental new information regarding sterol asymmetry in mammalian cell membranes will be obtained. At present no information regarding transbilayer distribution, function, and regulation of sterols in surface membranes of nucleated mammalian cells is available. The results of these investigations should be of value to our understanding of a number of disease states in which cellular sterol content is known to be abnormal. Membrane abnormalities in which transbilayer distribution of sterols are altered have not yet been identified and represent an additional area for future investigations.